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Biomedical subjects

L Rask

Publications and source records attributed to L Rask.

At least 73 records · Page 4Linked to original sources
PubMed1993

Cruciferin gene families are expressed coordinately but with tissue-specific differences during Brassica napus seed development.

The major storage protein in seeds of Brassica napus, the 12S globulin cruciferin, is composed of three different groups of subunits; cru1, cru2/3 and cru4. By using gene family-specific probes, we have investigated the accumulation, rate of synthesis and spatial distribution of transcripts corresponding to the different groups of cruciferin subunits in developing seeds. Cruciferin transcripts derived from different gene families accumulate coordinately to comparable amounts during seed development. The corresponding gene families are, however, transcribed at different rates. Investigation of the spatial distribution of transcripts corresponding to each group of cruciferin subunits in the developing seed by in situ hybridization, revealed that mRNAs of all three types accumulate in both axis and cotyledons. Transcripts derived from cru1 and cru4 gene families show a similar cell specificity and accumulate in a similar spatial manner during seed development. In contrast, mRNAs corresponding to the cru2/3 gene family are expressed with a partly different cell specificity and show a slightly different pattern of accumulation in the axis and cotyledons, with a delayed accumulation in epidermal cells. In the cotyledons, the initial accumulation of this type of cruciferin mRNAs is also distinguished from the two other types. The differences in cell specificity are seen in the root cap and in provascular cells, where mRNAs belonging to the cru2/3 family are absent.

Allergens↗
PubMed1993

Differential expression of myrosinase gene families.

In mature seeds of Brassica napus three major and three minor myrosinase isoenzymes were identified earlier. These myrosinases are known to be encoded by at least two different families of myrosinase genes, denoted MA and MB. In the work described in this paper the presence of different myrosinase isoenzymes in embryos, seedlings, and vegetative mature tissues of B. napus was studied and related to the expression of myrosinase MA and MB genes in the same tissues to facilitate future functional studies of these enzymes. In developing seeds, myrosinases of 75, 73, 70, 68, 66, and 65 kD were present. During seedling development there was a turnover of the myrosinase pool such that in 5-d-old seedlings the 75-, 70-, 66-, and 65-kD myrosinases were present, with the 70- and 75-kD myrosinases predominating. In 21-d-old seedlings the same myrosinases were present, but the 66- and 65-kD myrosinase species were most abundant. At flowering the mature organs of the plant contained only a 72-kD myrosinase. MA genes were expressed only in developing seeds, whereas MB genes were most highly expressed in seeds, seedling cotyledons, young leaves, and to a lesser extent other organs of the mature plant. During embryogenesis of B. napus, myrosinase MA and MB gene transcripts started to accumulate approximately 20 d after pollination and reached their highest level approximately 15 d later. MB transcripts accumulated to about 3 times the amount of MA transcripts. In situ hybridization analysis of B. napus embryos showed that MA transcripts were present predominatly in myrosin cells in the axis, whereas MB genes were expressed in myrosin cells of the entire embryo. The embryo axiz contained 75-, 70-, and 65-kD myrosinases, whereas the cotyledons contained mainly 70- and 65-kD myrosinases. Amino acid sequencing revealed the 75-kD myrosinase to be encoded by the MA gene family. The high degree of cell and tissue specificity of the expression of myrosinase genes suggests that studies of their transcription should provide interesting information concerning a complex type of gene regulation.

Amino Acid Sequence↗
PubMed1992

Ligand-dependent secretion of rat retinol-binding protein expressed in HeLa cells.

A minigene encoding rat retinol-binding protein (RBP) was transfected into HeLa cells, which do not express endogenous RBP, transthyretin, or cellular retinol-binding protein. The HeLa cells manufactured and secreted the transfected gene product, demonstrating that RBP-transthyretin assembly is not a requirement for the secretion of RBP. When HeLa cells were grown under vitamin A-deficient conditions, RBP accumulated in the endoplasmic reticulum. Both serum and retinol stimulated secretion of RBP in a concentration-dependent manner. The retinol-regulated secretion occurred also after protein synthesis had been blocked by cycloheximide. Addition of holo-RBP or retinal, but not retinoic acid, stimulated secretion of RBP. Thus, an in vitro model system that resembles the rat hepatocyte in vivo with regard to the known regulation of RBP secretion has been established in a human cell line of extrahepatic origin. It can be concluded that cellular retinol-binding protein is not required for the transfer of retinol to RBP and that the mechanism whereby retinol controls the intracellular transport of RBP is neither specific for tissues synthesizing RBP nor species-specific. To investigate the structural properties responsible for the endoplasmic reticulum retention of RBP in the absence of its ligand, a cDNA encoding chicken purpurin, a protein that is 50% identical to RBP and that binds retinol, was expressed in HeLa cells. In contrast to RBP, purpurin was not retained in vitamin A-deficient HeLa cells.

Animals↗
PubMed1992

A genetically engineered purpurin/retinol-binding protein hybrid that binds to transthyretin.

A mini-gene encoding rat retinol-binding protein (RBP) and a cDNA encoding chicken purpurin were separately transfected into HeLa cells. In contrast to RBP, expressed purpurin did not bind to transthyretin (TTR). A purpurin/RBP hybrid protein was constructed by substituting the cDNA sequence encoding the N-terminal 29 amino acids of purpurin for the corresponding part of RBP. The expressed hybrid molecule bound to the TTR-Sepharose. These results demonstrate that purpurin does not bind to TTR, that a functional purpurin/RBP hybrid can be constructed, and that the N-terminal coil of RBP is not required for TTR binding.

Amino Acid Sequence↗
PubMed1992

The glucosinolate-degrading enzyme myrosinase in Brassicaceae is encoded by a gene family.

A full-length cDNA clone (MB3) and three partial clones (MA1, MB1 and MB2) which encode myrosinase (thioglucoside glucohydrolase, EC 3.2.3.1) were isolated from a Sinapis alba (white mustard) cDNA library. Nucleotide sequence analysis of these clones revealed that they are encoded by a gene family. Southern blot analysis with gene-specific probes showed that the gene family consists of a least two subfamilies (MA and MB) each with several members both in S. alba and in Brassica napus (oilseed rape). In Arabidopsis thaliana (wall cress) only three myrosinase genes seem to be present. Northern blot analysis indicated that all the myrosinase mRNA species have the same size, approximately 1.95 kb.

Amino Acid Sequence↗
PubMed1992

Characterization of a Brassica napus gene encoding a cruciferin subunit: estimation of sizes of cruciferin gene families.

A gene encoding a subunit of the 12S storage globulin, cruciferin, in Brassica napus (oilseed rape) has been isolated and characterized. The gene consists of about 2200 bp including three short intervening sequences. Primer extension analysis showed that the major transcription start site is located 30 bp 5' of the predicted ATG start codon. This gene belongs to one of three different major families encoding cruciferin subunits. By use of gene-family-specific probes and Southern blotting analysis the number of genes of the three different cruciferin subtypes in B. napus was estimated.

Allergens↗
PubMed1992

Cloning of a cDNA for rape chloroplast 3-isopropylmalate dehydrogenase by genetic complementation in yeast.

Both insect and mammalian genes have previously been cloned by genetic complementation in yeast. In the present report, we show that the method can be applied also to plants. Thus, we have cloned a rape cDNA for 3-isopropylmalate dehydrogenase (IMDH) by complementation of a yeast leu2 mutation. The cDNA encodes a 52 kDA protein which has a putative chloroplast transit peptide. The in vitro made protein is imported into chloroplasts, concomitantly with a proteolytic cleavage. We conclude that the rape cDNA encodes a chloroplast IMDH. However, Southern analysis revealed that the corresponding gene is nuclear. In a comparison of IMDH sequences from various species, we found that the rape IMDH is more similar to bacterial than to eukaryotic proteins. This suggests that the rape gene could be of chloroplast origin, but has moved to the nucleus during evolution.

3-Isopropylmalate Dehydrogenase↗
PubMed1992

Purification and characterization of a surface lectin from the nematode-trapping fungus Arthrobotrys oligospora.

Several studies have indicated that the capture of nematodes by the nematophagous fungus Arthrobotrys oligospora is mediated by a lectin on the fungal surface. One of the major surface proteins of this fungus showed haemagglutinating activity and was isolated by affinity chromatography using a mucin Sepharose column. Biochemical analysis showed that the protein was a dimeric glycoprotein with a molecular mass of 36 kDa and an isoelectric point of pH 6.5, and contained no sulphur amino acids. The protein was N-terminally blocked; four internal peptides were sequenced, and showed no significant similarity to sequences in the Swiss-Prot or PIR databases. The haemagglutinating activity of the isolated protein was not inhibited by any of the mono- or disaccharides tested, but it was inhibited by the glycoproteins fetuin and mucin. The haemagglutinating activity changed after incubating the protein in buffers of different pH, with maximal activity at pH 11.0 and no activity at pH 2.8. The lectin was tested for different enzymic activities but none were detected. Analysis of the haemagglutinating activity in various cell fractions indicated that the protein was associated with extracellular polymer layers and with the cell wall of the fungus. About the same amount of the haemagglutinating protein was recovered from samples of vegetative mycelium and of mycelium containing nematode-trapping cells.

Amino Acid Sequence↗
PubMed1992

Distribution of napin and cruciferin in developing rape seed embryos.

The distribution of napin and cruciferin, the two major storage proteins in rape seed, Brassica napus, has been visualized during seed development by antibody staining of paraffin-embedded and sectioned seeds. The results indicate that the synthesis of both proteins during embryogenesis is strictly regulated with respect to time and tissue. Although the synthesis of napin started a few days earlier than that of cruciferin, both proteins displayed similar patterns in their spatial distributions. They were first detected in the axis, then in the outer cotyledon, and finally in the cells of the inner cotyledon. Both proteins are also present in the endosperm, although in lower amounts. In germinating seeds, napin and cruciferin were rapidly degraded. Within 2 days the amounts had decreased dramatically, and after 4 days hardly any cells contained napin or cruciferin. Biochemical analyses of dissected embryos showed that, for napin as well as for cruciferin, similar levels of polypeptides were found in the axis and cotyledons.

Journal Article↗
PubMed1991

cDNA cloning of the beta-subunit of the human gastric H,K-ATPase.

A full-length cDNA clone encoding the human gastric H,K-ATPase (EC 3.6.1.36)beta-subunit was isolated from a human gastric mucosal lambda gt10 library using oligonucleotide probes which were based on the cDNA sequence from rat and rabbit H,K-ATPase beta-subunits. The insert was 1407 bp in length and encoded a polypeptide of 291 amino acids with a MW = 33,367 Da. It exhibited 84.2%, 85.6% and 81.3% identity to the H,K-ATPase beta-subunits of rabbit, pig and rat, respectively.

Adenosine Triphosphatases↗
PubMed1991

Analysis of the promoter region of napin genes from Brassica napus demonstrates binding of nuclear protein in vitro to a conserved sequence motif.

Napin is a seed storage protein from Brassica napus (rape) that is encoded by a gene family. We have isolated and characterized a novel napin gene, napB. Comparisons of the 5'-upstream region of napB to the promoter regions of previously published napin genes reveal that certain sequence motives are evolutionary conserved and may be implicated in gene regulation. These consensus motives, that overlap with purine/pyrimidine stretches, are TACACAT and CATGCA both of which frequently occur as overlapping, direct repeats. Related or identical sequences are also found in the upstream regions of the homologous genes of Arabidopsis thaliana. One copy of the CATGCA motif occurs in close proximity to the TATA box in all the above genes. In this case it overlaps with an octamer sequence (ATGCAAAT) which is a sequence element common in many eukaryotic promoters and enhancers. The TACACAT sequence, as part of a longer purine/pyrimidine stretch, was found to interact with a protein present in crude nuclear extracts from developing B. napus seeds. Napin genes appear to be methylated to almost equal extents whether present in expressing or non-expressing tissue.

2S Albumins, Plant↗
PubMed1991

Characterization of the 12S globulin complex of Brassica napus. Evolutionary relationship to other 11-12S storage globulins.

Cruciferin (12S globulin) is the major seed protein in Brassica napus (oil seed rape). It is synthesized during seed development and consists of six subunit pairs. Each of these pairs is synthesized as a precursor containing one alpha and one beta chain. At least three different precursors exist (P1-3), giving rise to four different mature subunits (cru1-4). Several cruciferin clones were isolated from a seed mRNA cDNA library. Comparison of the deduced amino acid sequences of these clones to amino acid sequences of purified cruciferin chains and peptides identified them as coding for cru2/3 and cru4 subunits. From the amino acid sequences deduced from two overlapping cDNA clones, the precursor of the cru4 subunit was shown to consist of 465 amino acid residues. Comparison of cruciferin and cruciferin-related sequences from B. napus and Arabidopsis thaliana, respectively, suggested that early during evolution the Brassicaceae family only possessed two types of 11-12S globulin genes, like the present-day Fabaceae.

Allergens↗
PubMed1991

Concerted evolution in a segment of the first domain exon of polymorphic MHC class II beta loci.

Genetic exchange of sequence information between members of a gene family, generally denoted gene conversion, causes a phenomenon called "concerted evolution" meaning that non-allelic genes do not evolve independently. The possible significance of this phenomenon in the evolution of major histocompatibility complex (MHC) class II genes has been investigated in the present study. The results of a phylogenetic analysis of human, mouse, bovine, and chicken class II sequences were consistent with the occurrence of gene conversion between polymorphic class II beta genes (i.e. DPB, DQB, and DRB) but not between these genes and the monomorphic DOB gene or between class II alpha genes. Gene conversion between polymorphic beta genes appears to be restricted to a gene segment between approximately nucleotide positions 94-286 in the first domain exon. Due to this genetic exchange, there is a greater interlocus similarity both at the DNA and protein level in this region than in the rest of the sequence. The region encodes a functionally important part of the class II molecule including more than half of the beta-chain residues of the antigen binding site and the residues in the alpha helix assumed to form contact with the T-cell receptor. The observed similarity in the alpha-helical region of class II beta molecules may be functionally significant for the utilization of the T-cell repertoire for antigen recognition in the immune system.

Animals↗
PubMed1991

Retinol-binding protein and transthyretin expressed in HeLa cells form a complex in the endoplasmic reticulum in both the absence and the presence of retinol.

To establish a suitable experimental system for studies of the interaction of retinol-binding protein (RBP) with transthyretin (TTR) we have expressed the corresponding cDNAs in HeLa cells. To investigate whether complex formation might occur already in the endoplasmic reticulum (ER), the C-terminal ER retention signal, KDEL, was attached to TTR. The tetrameric TTR-KDEL fusion protein was retained in the ER of HeLa cells. When RBP was co-expressed with TTR-KDEL, RBP was retained intracellularly. A cDNA-encoding purpurin, a protein which is 50% identical to RBP, was then expressed together with TTR-KDEL. Purpurin was not retained intracellularly and did not bind to TTR coupled to Sepharose. The effect of the vitamin A status on the secretion of TTR and RBP was examined. While TTR expressed alone was not retained intracellularly, TTR was retained in vitamin A-deficient cells when co-expressed with RBP. Addition of retinol stimulated rapid secretion of both proteins. These results demonstrate that TTR can form a complex with RBP in the ER. The data suggest that RBP and TTR are secreted as a complex.

Anthraquinones↗
PubMed1991

Distribution of myrosinase in rapeseed tissues.

Immunocytochemical studies on Brassica napus (rapeseed) tissues using a monoclonal antibody against myrosinase (thioglucoside glucohydrolase) showed that the enzyme was only present in a small number of cells. In the developing embryo, scattered myrosinase-containing cells were present in both cotyledons and axis. The enzyme accumulated in these cells during the later stages of seed development, approximately from day 20 until day 40 after pollination. Parallel staining with the immunocytochemical technique and a histochemical method identified these cells as myrosin cells. Myrosinase appeared to be located outside the myrosin grains, although the occasional association with the membrane of the grains also was noted. In leaves, petals, and siliques, scattered parenchyma cells were stained in the mesophyll as well as in the vascular tissue. In young leaves, guard cells also contained myrosinase. The enzyme was also present in xylem cells of the stem.

Journal Article↗